Therapeutic compositions

ABSTRACT

Compositions comprising ketone bodies and/or their metabolic precursors are provided that are suitable for administration to humans and animals and which have the properties of, inter alia, (i) increasing cardiac efficiency, particularly efficiency in use of glucose, (ii) for providing energy source, particularly in diabetes and insulin resistant states and (iii) treating disorders caused by damage to brain cells, particularly by retarding or preventing brain damage in memory associated brain areas such as found in Alzheimer&#39;s and similar conditions.  
     These compositions may be taken as nutritional aids, for example for athletes, or for the treatment of medical conditions, particularly those associated with poor cardiac efficiency, insulin resistance and neuronal damage. The invention further provides methods of treatment and novel esters and polymers for inclusion in the compositions of the invention.

[0001] The present invention relates to compositions suitable foradministration to humans and animals which have the properties of, interalia, (i) increasing cardiac efficiency, particularly efficiency in useof glucose, (ii) for providing energy source, particularly in diabetesand insulin resistant states and (iii) treating disorders caused bydamage to brain cells, particularly by retarding or preventing braindamage in memory associated brain areas such as found in Alzheimer's andsimilar conditions. These compositions may be taken as nutritional aids,for example for athletes, or for the treatment of medical conditions,particularly those associated with poor cardiac efficiency, insulinresistance and memory loss. The invention further provides methods oftreatment and novel esters and polymers for inclusion in thecompositions of the invention.

[0002] Abnormal elevation of blood sugar occurs not only in insulindeficient and non insulin dependent diabetes but also in a variety ofother diseases. The hyperglycaemia of diabetes results from an inabilityto metabolize and the over production of glucose. Both types of diabetesare treated with diet; Type I diabetes almost always requires additionalinsulin, whereas non-insulin dependent diabetes, such as senile onsetdiabetes, may be treated with diet and weight loss, although insulin isincreasingly used to control hyperglycaemia.

[0003] Increased sympathetic stimulation or elevated glucagon levels, inaddition to increasing glycogenolysis in liver, also stimulate freefatty acid release from adipocytes. After acute myocardial infarction orduring heart failure, increased sympathetic nervous activity oradministration of sympathomimetics accelerate glycogenolysis, decreaserelease of insulin from P cells of the pancreas and cause relativeinsulin resistance. While the importance of diet, or substrateavailability, is taken as a given in the treatment of diabetes, thecritical effects of substrate choice in insulin resistant states has notbeen widely appreciated or applied in clinical practice. Insteadcontemporary interest has focused upon the complex signalling cascadewhich follows the binding of insulin to its receptor. This increasinglycomplex cascade of messages involving protein tyrosine kinases andphosphatases, inositol and other phospholipids, while holding promisefor the ultimate understanding of non-insulin dependent diabetes, hasyet to provide significant new therapies for either diabetes or insulinresistance.

[0004] Leaving aside the longer term effects of insulin on growth, theacute metabolic effects of insulin have been thought to be accounted forby action at three major enzymatic steps in the conversion of glucose toCO₂. Firstly insulin promotes the translocation of the glucosetransporter, Glut4, from endoplasmic reticular to plasma membranes, thusincreasing the transport of glucose from the extra to intracellularphase (see refs. 1 and 2). Secondly, insulin increases the accumulationof glycogen. This has been attributed to dephosphorylation of glycogensynthase (3) by protein phosphatase 1. Thirdly, insulin stimulates theactivity of mitochondrial pyruvate dehydrogenase multi-enzyme complex (4and 5) through dephosphorylation by a Ca²⁺ sensitive (6)intramitochondrial protein phosphosphatase.

[0005] An important, but poorly understood effect of insulin is its usein cardiac disease where in combination with glucose, potassium chlorideand GIK, it improved electrocardiographic abnormalities accompanyingmyocardial infarction (7 and 8), and improved cardiac performance afterpost pump stunning (9). This treatment has been advocated recently for anumber of other serious cardiac diseases (10 and 11). The beneficialeffects of GIK infusion have been attributed to its ability to decreasefree fatty acid release and improve membrane stability (12). However,other more recent work suggests more fundamental reasons. In heart cellsthat are anoxic, glucose is the only fuel capable of providing the ATPnecessary to maintain viability (13).

[0006] Administration of glucose plus insulin would increase theavailability of intracellular glucose providing a source of ATPproduction in the absence of O₂. While this would explain certainbeneficial effects, it would not account for the correction of EKGabnormalities nor the improved cardiac index in hearts treated with GIKbecause electrical activity and cardiac work requires actively respiringcardiac cells, not ones which are totally anoxic and therefore withoutelectrical activity or the ability to perform mechanical work.

[0007] Understanding the enzymatic sites of insulin's action does not,by itself, define the effects of insulin deficiency upon the cellularmetabolism or physiological function. How insulin acts at this largerlevel can best be understood by looking at the way nature deals withinsulin deficiency. The natural compensation for decreased insulinduring fasting is the accelerated hepatic conversion of the free fattyacids to the ketone bodies raising blood D-β-hydroxybutyrate andacetoacetate to about 6 mM. At these levels, ketones, rather thanglucose, become the substrate for most organs, including even the brain(14). Although mild ketosis is the normal response to decreased insulin,physicians fear ketone bodies because their massive overproduction canbe life threatening in diabetic ketoacidosis.

[0008] The present inventor has previously compared the effect ofphysiological levels of ketone bodies to the metabolic and physiologicaleffects of insulin, particularly comparing the insulin deficient workingrat heart perfused with glucose alone, to hearts to which was addedeither 4 mM D-β-hydroxybutyrate/1 mM acetoacetate, saturating doses ofinsulin or the combination and has shown how provision of simplesubstrates can mimic the effects of insulin in changing theconcentrations of the intermediates of both glycolysis and the TCA cycleand thereby controlling the flux of glucose in this very specialisedtissue. In addition he has determined that a primary but previouslyunrecognized effect of insulin or a ratio of ketones is to altermitochondrial redox states in such a way so as to increase theΔG_(ATPhydrolysis) and with that, the gradients of inorganic ionsbetween the various cellular phases and the physiological performance ofheart.

[0009] The present application teaches that such ketone bodies can alsoprovide a therapeutic approach to the treatment of insulin resistancewhere the normal insulin signalling pathway is disordered and inconditions where the efficiency of cardiac hydraulic work is decreasedfor metabolic reasons. The inventor has determined that use of ketonebodies has great advantage over use of insulin itself for reasons thatwill become evident from the description below, not least of these beingthe elimination of carbohydrate intake control otherwise necessary.

[0010] The present application further addresses the problem ofneurodegenerative diseases, particularly disease where neurons aresubject to neurotoxic effects of pathogenic agents such as proteinplaques and further provides compositions for use in treating these andthe aforesaid disorders.

[0011] Alzheimer's disease is a genetically heterogeneous group ofprogressively fatal neurological diseases characterized pathologicallyby accumulation of amyloid plaques in brain and clinically by impairmentof recent memory leading to dementia and death. In addition to the casesof Alzheimer's disease linked to genetic causes, sporadic cases, withoutan apparent family history of the disease, also occur. For examplepathological changes characteristic of Alzheimer's disease occur afterhead trauma (73) or after inflammatory diseases stimulating productionof the cytokine interleukin-1 (97).

[0012] The early symptom of the disease is loss of recent memoryassociated with impairment and death of cell in the hippocampusaccounting for the early impairment of recent memory. Measurement of thehippocampal volumes using magnetic resonance imaging (MRI) shows thatatrophy of hippocampus occurs prior to the clinical onset of memory lossand procresses with a loss of volume of about 8% per year during the 2years over which symptoms first appeared (70).

[0013] The diagnosis of Alzheimer's disease is made clinically by thisimpairment in recent memory, associated with lesions in the hippocampalportion of the temporal lobe. Neuropathologically, the diagnosis dependsupon the finding of neurofibrillatory tangles within the cells, amyloidor senile plaques in the extracellular space and loss of neuronal number(61). The neurofibrillatory tangles are comprised of pairedhyperphosphorylated tau protein, whose usual function in the cell, whennot phosphorylated, is to bind to and stabilize tubulin in its formationof microtubules within the cell. Hyperphosphorylation of tau iscatalysed by glycogen synthase kinase 3β, among other kinases anddephosphorylated by protein phosphatase 2A-1, 2B or 1 (108).

[0014] However, there is not necessarily a clear, bright line betweenthe pathological brain changes and the memory deficits which occurprematurely in Alzheimer's disease and the pathological changes in brainanatomy and memory function which are found in the “normal” agingpopulation. Rather the difference is a quantitative one dependent uponrate (94). Such changes in memory function in the normal aged are alsoaccompanied by a decreased glucose tolerance signifying an inability tometabolize glucose. In such situations, treatments aimed at rectifyingthe pathophysiological processes of Alzheimer's disease, would beexpected to be applicable to the correction of the metabolic effectsassociated with normal aging.

[0015] While Alzheimer's disease of the familial or the sporadic type isthe major dementia found in the aging population, other types ofdementia are also found. These include but are not limited to: thefronto-temporal degeneration associated with Pick's disease, vasculardementia, senile dementia of Lewy body type, dementia of Parkinsonismwith frontal atrophy, progressive supranuclear palsy and corticobasaldegeneration and Downs syndrome associated Alzheimers. Plaque formationis also seen in the spongiform encephalopathies such as CJD, scrapie andBSE. The present invention is directed to treatment of suchneurodegenerative diseases, particularly those involving neurotoxicprotein plaques, eg. amyloid plaques.

[0016] Many of these aforesaid apparently unrelated conditions have thehyperphosphorylated tau proteins found in Alzheimer's disease (69),opening up the possibility that the same kinase which phosphorylated tauwould also phosphorylate the PDH complex producing a similar deficiencyin mitochondrial energy production and acetyl choline synthesis found inAlzheimer's disease but involving other brain regions. The presentinventor has determined that in this respect treatments applicable toAlzheimer's disease might be applied to these diseases as well. Inaddition, the inventor has determined that such treatment will also beapplicable to peripheral neurological wasting diseases, such asmyasthenia gravis and muscular dystrophy.

[0017] At present there is no effective treatment for Alzheimer'sdisease. Research efforts are focused on defining its genetic cause butto date there has been no succesful gene therapy. Genetic studies havelinked Alzheimer's disease with Mongolism and in its early onset form tolocus on chromosome 21 causing accumulation of amyloid precursor protein(APP)(73), a transmembrane glycoprotein existing in 8 isoforms. Numerousfragments of this protein are derived by proteolysis and the plaquescharacteristic of Alzheimer's disease have been shown to containaccumulation of the oligomer of β amyloid protein (A β₁₋₄₂). An earlyonset autosomally dominant form of Alzheimer's disease has also beenrelated to a presenilin 1 locus on chromosome 14.

[0018] A late onset form of Alzheimer's disease is associated with thetype 4 allele of apolipoprotein E (69,98) on chromosome 19, althoughother workers suggest that this apparent correlation may be relatedinstead of α 1 antichymotrypsin locus instead (100). All transgenic miceexpressing increased amounts of amyloid precursor protein over 18 monthsof age showed hippocampal degeneration with many of the pathologicalcharacteristics of Alzheimer's disease (90).

[0019] The current status of knowledge on the defective genes and geneproducts in Alzheimer's disease has recently been summarized (Table 1 ofref. 96). Chro- mo- some Gene Defect Age of Onset Aβ Phenotype 21 βAPPmutations 50's {circumflex over ( )} Production of total Aβ peptides ofAβ₁₋₁₂ 19 apoE4 polymorphism 60's or > {circumflex over ( )} density ofAβ plaques and vascular deposits 14 Presenilin 1 mutations 40's & 50's{circumflex over ( )} production of Aβ₁₋₁₂  1 Presenilin 2 mutations50's {circumflex over ( )} production of Aβ₁₋₁₂

[0020] It is clear from the above table that the common phenotypeassociated with the genetic forms of Alzheimer's disease is theaccumulation of the amyloid peptide Aβ₁₋₄₂ (96). It is this Aβ₁₋₄₂ whichinactivates PDH thus impairing mitochondrial energy and citrateproduction in normally obligate glucose consuming tissue (95) and at thesame time impairing synthesis of the critical neurotransmitter, acetylcholine (67,68). The application of Aβ₁₋₄₂ to neuronal cells isassociated with the downregulation of the anti-apototic protein bcl-1and increases levels of bax, a protein known to be associated with celldeath (92). In addition to amyloid plaques comprised of Aβ₁₋₄₂,neurofibrillatory tangles comprised of hyperphosphorylated tau protein,and decreased brain acetyl choline levels, cell death is the fourthpathological characteristic of Alzheimer's disease. These pathologicalcharacteristics can be related, at least in part, to excess Aβ₁₋₄₂ andits inhibition of PDH.

[0021] Modest clinical improvement in symptoms can occur by treatmentwith acetyl choline esterase inhibitors (57), presumably by increasingcholinergic efferents originating in the septal nuclei and traversingBroca's diagonal band to hippocampus in the anterior portion of thelimbic system of brain. However the progress in the molecular biology ofAlzheimer's disease has caused the search for new therapies toconcentrate upon four major areas (96): (i) protease inhibitors thatpartially decrease the activity of the enzymes (β and γ secretase) thatcleave Aβ (β amyloid fragments) from βAPP (β amyloid precursorproteins); (ii) compounds that bind to extracellular Aβ that prevent itscytotoxic effects; (iii) brain specific anti-inflammatory drugs thatblock the microglial (brain macrophages) activation, cytokine release,and acute phase response that occur in affected brain regions; and (iv)compounds such as antioxidants, neuronal calcium channel blocks, orantiapoptotic agents that interfere with the mechanisms of Aβ triggeredneurotoxicity.

[0022] The therapy which the present inventor now proposes differs fromthe four approaches listed above in that it bypasses the block inmetabolic energy production resulting from inhibition of PDH by Aβ₁₋₄₂by administering ketone bodies or their precursors. Neuronal cells arecapable of metabolizing such compounds even in the presence of adeficiency of glucose, the normal energy substrate for brain (63).Because ketones can increase the ΔG of ATP hydrolysis, the gradients ofboth intracellular Na⁺ and Ca²⁺ will be increased, preventing cell deathassociated with increased intracellular Ca2+. Furthermore, the increasein citrate generation by the Krebs cycle will provide, when translocatedinto cytoplasm, a source of cytoplasmic acetyl CoA required to remedythe deficiency of acetyl choline characteristic of Alzheimer's brains.

[0023] The elevation of blood ketones necessary to correct thesemetabolic defects can be accomplished by parenteral, enteral means ordietary means and does not require the administration of potentiallytoxic pharmacological agents.

[0024] There has been long experience with ketogenic diets in childrentreated for epilepsy. Such diets are however unsuitable for use inadults due to adverse effects on the circulatory system. The presentinventions application of ketone bodies should provide all thetherapeutic effects of such diet, which is not itself found to be toxicin children, with none of the side effects that render it unused adults.Furthermore, the inventor has determined that with the correction of theaforesaid metabolic defects, cytokine responses and the increase inapoptotic peptides in degenerating cells will decrease due to theincrease in neuronal cell energy status and the increased trophicstimulation resulting from increased acetyl choline synthesis.

[0025] Since the priority date of this application, EP 0780123 A1 hasbeen published which relates to use of acetoacetate, β-hydroxybutyrate,monhydric, dihydric or trihydric alcohol esters of these or oligomers ofβ-hydroxybutyrate for suppressing cerebral edema, protecting cerebralfunction, rectifying cerebral energy metabolism and reducing the extentof cerebral infarction. It should be noted however, that it has beenknown since 1979 that sodium hydroxybutyrate increases cerebralcirculation and regional vasomotor reflexes by up to 40%(Biull.Eksp.Biol.Med Vol 88 11, pp555-557). The treatment that thepresent inventor now provides goes beyond such effects on circulation asit provides treatment for cells that are unable to function due toneurodegeneration, eg caused by neurotoxic agents such as peptides andproteins, and genetic abnormality. The treatment involves action ofketone bodies on the cells themselves and not the flow of blood to them.

[0026] In reducing this invention to practice the inventor has furtherdetermined that ketone bodies, provided by direct adminsitration or byadministration of their metabolic precursors in amounts sufficient toraise total blood ketone body concentration to elevated levels result inmore than simple maintenance of cell viability but actually improve cellfunction and growth beyond that of normal, ie. control levels in amanner unrelated to blood flow or nutrition. In this respect theinvention further provides use of ketone bodies as nerve stimulantfactors, ie. nerve growth factors and factors capable of stimulatingenhanced neuronal function, such as increase of metabolic rate andincrease of extent of functional features such as axons and dendrites.This aspect of the present invention offers a mechanism for improvementof neuronal function as well as mere retardation of degredation.

[0027] The recent work of Hoshi and collaborators (77, 78) stronglysuggests that a part of the amyloid protein whose accumulation is thehallmark of Alzheimer's disease, Aβ₁₋₄₂, acts as a mitochondrialhistidine protein kinase which phosphorylates and inactivates thepyruvate dehydrogenase multienzyme complex. The PDH complex is amitochondrial enzyme responsible for the generation of acetyl CoA andNADH from the pyruvate produced by glycolysis within the cytoplasm. Themitochondrial acetyl CoA formed condenses with oxaloacetate to start theKrebs TCA cycle completely combusting pyruvate to CO₂ while providingthe mitochondria with the reducing power which becomes the substrate forthe electron transport system through which the energy required formitochondrial ATP synthesis is generated. PDH thus stands at thecrossroads of the two major energy producing pathways of the cell,glycolysis and the Krebs cycle, and clearly serves a critical functionin living cells.

[0028] There are two major consequences of the inhibition of PDH.Firstly, in neuronal tissues, which under normal metabolic conditionsare totally dependent upon glucose for energy production, inhibition ofPDH results in a lowered efficiency of energy production, a loweredenergy of hydrolysis of ATP, a decrease in both acetyl CoA and themetabolites of the first ⅓ of the TCA cycle and a deficiency ofmitochondrial NADH (95). A decrease in the energy of ATP hydrolysisleads to increased intracellular Na⁺ and Ca²⁺, loss of cellular K⁺ andultimately cell death (86). Hippocampal cells, critical for the fixationof recent memories, are particularly sensitive to a number of forms ofinjury, and the death of these cells is the hallmark both clinically andpathologically of Alzheimer's disease.

[0029] A second major consequence of PDH inhibition is a deficiency ofmitochondrial citrate (95). Citrate, or one of its metabolites, isexported to the cytoplasm from mitochondria where it is converted tocytosolic acetyl CoA by ATP citrate lyase (EC 4.1.3.8) in the reaction:

citrate³⁻+ATP⁴⁻+CoASH>acetyl CoA+oxaloacetate²⁻+ADP³⁻+HPO₄ ²⁻

[0030] The acetyl CoA then combines with choline through the action ofcholine acetyl transferase (EC 2.3.1.6) to form acetyl choline in thereaction:

choline⁺+acetyl CoA>CoASH+acetyl choline⁺

[0031] Neuronal culture of septal cells exposed to 1 μm Aβ₁₋₄₂ for 24hours showed a decrease in acetyl choline production of over five fold(78) with no decrease in the activity of choline acetyl transferase. Theinferred cause of this decreased production was a deficiency of acetylCoA due to inhibition of the PDH complex caused by activation of theTPKI/GSK-3β protein kinase and subsequent phosphorylation of PDH (77).

[0032] As explained above isolated working hearts perfused with 10 mMglucose alone without insulin are inefficient and have impairedmitochondnral energy production. This defect in cellular energyproduction can be completely reversed by the provision of aphysiological ratio of ketone bodies consisting of 4 mM D-βhydroxybutyrate and 1 mM acetoacetate (95). Brain was thought to becapable of using only glucose as its metabolic energy source and to beinsensitive to the actions of insulin. However, in a remarkable clinicalstudy performed in 1967, George Cahill and his collaborators (47) showedthat up to 60% of the brain's need for metabolic energy could be met byketone bodies in obese patients undergoing prolonged fasting. Even moreremarkably, Cahill showed that administration of insulin to thesepatients in doses sufficient to drop their blood sugar from 4 to under 2mM was associated with no impairment of mental functions in thesepatients whose blood D-β hydroxybutyrate was 5.5 mM and acetoacetate 2mM (see FIG. 3 from ref 63). Clearly, when ketone bodies are present inthe blood at levels above 5 mM, they are able to substitute for thebrain's usual need for glucose and abolish the hypoglycemic symptomsexpected at blood glucose levels of 1.5 mM

[0033] Ketone body utilization in brain is limited by the transport,with lesser utilization occurring in the basal ganglion at blood levelsbelow 1 mM (76). However, at levels of 7.5 mM achieved in normal man byprolonged fasting, the rate of ketone body entry into brain issufficient to take over the majority of cerebral energy needs and toprevent hypoglycemic symptoms, even in the face of blood sugar levelswhich would normally cause convulsions or coma (63).

[0034] It is the inventors hypothesis that in Alzheimer's disease, wherethere is a block at PDH which prevents the normal energy production fromglucose, if one can provide elevated, eg. normal fasting levels ofketones, one can bypass the PDH blockade present in these patientsthereby preventing cell death due to energy depletion or lack ofcholinergic stimulation and thus slow the progression of the memory lossand dementia.

[0035] Furthermore, utilising the nerve growth/stimulatory effects ofthe ketone bodies, particularly D-β-hydroxybutyrate or a physiologicalratio of this with acetoacetate, cells that are still viable can becaused to improve beyond the state to which they have degenerated andaccordingly some improvement of function will be seen in patients.

[0036] In fed animals and in man the liver content, which is essentiallythat of blood, of acetoacetate is very low at 0.09 mM and D-βhydroxybutyrate is 0.123 mM but rises after a 48 hour fast to 0.65 mMacetoacetate and 1.8 mM D-β hydroxybutyrate (84). The ketone bodies risein starvation because the fall in insulin decreases there-esterification of fatty acids to triglyceride in adipose tissuecausing the release of free fatty acids into the blood stream. Thereleased free fatty acids can then be taken up and used as a source ofenergy by muscle, heart, kidney and liver in the process of β oxidation.Liver, however, has the capacity to convert the free fatty acids to ametabolic fuel, ketones, for use by extrahepatic organs, including thebrain, as an alternative to glucose during periods of fasting. Thehepatic synthesis of ketone bodies occurs from mitochondrial acetyl CoAgenerated during the β-oxidation of fatty acids by liver in thefollowing set of reactions:

[0037] Once made in the liver, ketone bodies are transported out of theliver into the blood stream by the monocarboxylate-H′ co-transporter(20) by the following reaction:

[0038] The ketone bodies enter extra-hepatic tissues on the samecarrier, where other monocarboxylates can act as competitive inhibitors.Unphysiological isomers such as D-lactate or L-β-hydroxybutyrate canalso act as competitive inhibitors to ketone body transport. Sinceketone body transport across the blood brain barrier is the limitingfactor to ketone body utilization in brain (76) every effort should bemade to keep the blood concentration of these unphysiologicalenantiomers at low levels during ketogenic therapy. When blood ketonebody concentrations are elevated to levels found in starvation, heart,muscle, kidney and brain utilize ketone bodies as the preferred energysubstrate:

 Citrate³⁻→Krebs TCA cycle

[0039] The present inventor has thus determined that the mitochondrialacetyl CoA from ketone bodies can thus replace the acetyl CoA deficiencywhich occurs during inhibition of PDH multienzyme complex in tissuesdependent upon the metabolism of glucose for their supply of metabolicenergy. The mitochondrial citrate supplied can also be transported tocytoplasm by the tri or dicarboxcylic acid transporter where it can beconverted to cytoplasmic acetyl CoA required for the synthesis of acetylcholine. The reactions of the Krebs cycle are shown in Scheme 1 to helpillustrate these concepts further.

[0040] The liver cannot utilize ketone bodies because it lacks the 3Oxoacid CoA transferase necessary for the formation of acetoacetyl CoA.Ketone bodies, in contrast to free fatty acids, cannot produce acetylCoA in liver. Since acetyl CoA is the essential precursor of fatty acidsynthesis through malonyl CoA and cholesterol synthesis throughcytosolic HMG CoA, ketone bodies cannot result in either increased fattyacid or cholesterol synthesis in liver, which usually accounts for overhalf of the bodies synthesis of these two potentially pathogenicmaterials. Liver is sensitive to the ratio ofactoacetate/D-β-hydroxybutyrate presented to it and will alter itsmitochondrial free [NAD⁻]/[NADH], because of the near equilibriumestablished by β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) (55)

[0041] The easiest way to increase blood ketones is starvation. Onprolonged fasting blood ketones reach levels of 7.5 mM (62, 63).However, this option is not available on a long term basis, since deathroutinely occurs after a 60 day fast.

[0042] The ketogenic diet, comprised mainly of lipid, has been usedsince 1921 for the treatment of epilepsy in children, particularlymyoclonic and a kinetic seizures (109) and has proven effective in casesrefractory to usual pharmacological means (71). Either oral orparenteral administration of free fatty acids or triglycerides canincrease blood ketones, provided carbohydrate and insulin are low toprevent re-esterification in adipose tissue. Rats fed diets comprised of70% corn oil, 20% casein hydrolysate, 5% cellulose, 5% McCollums saltmixture, develop blood ketones of about 2 mM. Substitution of lard forcorn oil raises blood ketones to almost 5 mM (Veech, unpublished).

[0043] An example of a traditional 1500/day calorie ketogenic dietrecommended by the Marriott Corp. Health Care Services, Pediatric DietManual, Revised August 1987 as suitable for a 4-6 year old epilepticchild contained from 3:1 to 4:1 g of fat for each g of combinedcarbohydrate and protein. At each of 3 meals the patient must eat 48 to50 g fat, only 6 g protein and 10 to 6.5 g carbohydrate. In practicethis means that at each meal the child must eat 32 g of margarine perday (about ¼ stick) and drink 92 g of heavy cream (about 100 ml),comprised mainly as medium chain length triglycerides.

[0044] An example of a diet achieving a 3:1 ratio of fat to combinedcarbohydrate and protein is given in Table 1 below. TABLE 1 Sample 1500calorie diet to achieve 3:1 lipid to carbohydrate + protein diet Amount(g) Fat (g) Protein (g) CHO (g) Breakfast Egg 32 4 4 apple juice 70 7margarine 11 10 heavy cream 92 34 2 3 Total Breakfast 48 6 10 Lunch leanbeef 12 1.75 3.5 cooked carrots 45 0.6 3 canned pears 40 4 margarine 1412.5 heavy cream 92 34 2 3 Total Lunch 48.25 6.1 10 Supper Frankfurter22.5 6 3 Cooked broccoli 50 1 2 Watermelon 75 5 Margarine 8 7.5 Heavycream 92 34 2 3 Total Supper 47.5 6 10 Daily Total 143.75 18.1 30

[0045] In general the levels of ketone bodies achieved on such diets,are about 2 mM D-β hydroxybutyrate and 1 mM acetoacetate while thelevels of free fatty acids about 1 mM. Other variations of compositionhave been tried including medium chain length triglycerides. In generalcompliance with such restricted diets has been poor because of theirunpalatability (56). High lipid, low carbohydrate diets also have beentried as therapeutic agents in cancer patients to reduce glucoseavailability to tumors (88) as weight reducing diets in patients withand without diabetes (74, 112) to improve exercise tolerance (83).

[0046] The limitation of diets which rely upon lipid to raise bloodketones to neurologically effective levels are many. Firstly, levels ofketone bodies on lipid based diets tend to be below 3 mM, significantlylower than the level of 7.5 mM achieved in normal obese humans duringprolonged fasting. Secondly, unauthorized ingestion of carbohydrateincreases insulin secretion and causes a rapid decrease in the hepaticconversion of free fatty acids to ketones with a consequent drop inblood ketones and the diversion of lipid to esterified to triglyceridesby adipose tissue. Many anecdotal reports relate the resumption ofseizures in children who “broke their diet with birthday cake”. Thirdlythe unpalatability and the necessity to avoid carbohydrate to sustainhigh ketone body levels makes such high lipid diets difficult to use inadults in an out patient setting, particularly in societies wheretraditionally high intake of refined sugars, bread, pasta, rice andpotatoes occurs. In practice, the traditional high ketone diet cannot beenforced in patients, other than children beyond the age where all foodis prepared at home under strict supervision. Fourthly, ingestion ofsuch large amounts of lipid in the adult population would lead tosignificant hypertriglyceridemia with its pathological sequelae ofincreased vascular disease and sporadic hepatic and pancreatic disease,and therefore could not be prescribed on medical grounds. Ingestion ofhigh lipid, low carbohydrate diets were popular in the 1970s for weightreduction in the face of high caloric intake, provided that carbohydrateintake was low. However, because of the increased awareness of therelationship of elevated blood lipids to atherosclerosis the popularityof this diet dropped abruptly.

[0047] Supplementing a liquid diet with 47% of its caloric content witheither glucose or racemic 1,3 butandiol caused the blood ketoneconcentration to rise about 10 fold to 0.98 mM D-β hydroxybutyrate and0.33 mM acetoacetate (107). These values are slightly less than obtainednormally in a 48 hour fast and far below the levels of 7.5 mM obtainedin fasting man. Racemic 1,3 butandiol is converted by liver toacetoacetate and both the unnatural L-β and the natural D-βhydroxybutyrate (respectively (S) 3-hydroxybutanoate and (R)3-hydroxybutanoate). Although racemic 1,3 butandiol has been extensivelystudied as a cheap caloric source in animal food and has even been usedexperimentally in human diets (81, 101) the production of the unnaturalL-isomer is likely in the long run to produce significant toxicity ashas been shown for the human use of the unnatural D-lactate (64). Onedisadvantage of administering the unnatural L isomer is that it competesfor transport with the natural D-β hydroxybutyrate. Thus provision ofthe (R) 1,3 butandiol as a precursor of ketone bodies is one possibilitythat avoids unnecessary administration or production of the unnaturalisomer.

[0048] The mono and diester of racemic 1,3 butandiol have been suggestedas a source of calories and tested in pigs (67). Oral administration ofa bolus of a diet containing 30% of calories as the esters producedbrief peaks blood ketones to 5 mM. However, the use of racemic 1,3butandiol with its production of the abnormal (S) 3-hydroxybutanoate isnot to be recommended for the reasons stated above.

[0049] While use of racemic 1,3 butandiol in such formulations is notrecommended, the esters of (R) 1,3 butandiol can be used, either aloneor as the acetoacetate ester. (R) 1,3 butandiol may easily besynthesized by reduction of the monomeric D-β hydroxybutyrate, with forexample LiAlH₄. (R) 1,3 butandiol is subject to being oxidized in theliver to form D-β hydroxybutyrate without marked distortion of thehepatic redox state. Studies in rats have shown that feeding racemic 1,3butandiol caused liver cytosolic [NAD′]/[NADH] to decrease from 1500 toabout 1000 (87). By comparison, administration of ethanol reduceshepatic [NAD−]/[NADH] to around 200 (106).

[0050] Acetoacetate, when freshly prepared, can be used in infusionsolutions where it can be given in physiologically normal ratios tooptimum effect (95). Because of manufacturing requirements whichcurrently require long shelf life and heat sterilized fluids,acetoacetate has frequently been given in the form of an ester. This hasbeen done to increase its shelf life and increase its stability to heatduring sterilization. In the blood stream, esterase activity has beenestimated to be about 0.1 mmol/min/ml and in liver about 15 mmol/min/g(68). In addition to esters combining 1,3 butandiol and acetoacetatethere has also been extensive study of glycerol esters of acetoacetatein parenteral (59) and enteral nutrition (82). Such preparations werereported to decrease gut atrophy, due to the high uptake of acetoacetateby gut cells and to be useful in treatment of burns (85).

[0051] However, neither 1,3 butandiol, which forms acetoacetate, norglycerol, which is a precursor of glucose, is part of the normal redoxcouple, D-β hydroxybutyrate/acetoacetate. For the present invention,under optimum conditions, a physiological ratio of ketones should begiven. If it is not, in the whole animal, the liver will adjust theratio of ketones in accordance with its own mitochondrial free[NAD⁺]/[NADH]. If an abnormal ratio of ketones is given pathologicalconsequences are a distinct possibility. In the working heart, perfusionwith acetoacetate as sole substrate, rapidly induces heart failure (99)in contrast to rat hearts perfused with a mixture of glucose,acetoacetate and D-β hydroxybutyrate, where cardiac efficiency wasincreased by a physiological ratio of ketone bodies (95).

[0052] The best exogenous source of ketone bodies, which do not requireingestion of large amounts of lipid nor the use of material whichproduce the physiologically incompatible isomers L-β-hydroxybutyratewould be ketone bodies themselves. However the present invention alsoprovides alternatives for administration in therapy.

[0053] A first alternative are polyesters of D-β-hydroxybutyrate.Natural polyesters of D-β-hydroxybutyrate are sold as articles ofcommerce at polymers of 530,000 MW from Alcaligenes eutrophus (SigmaChemical Co. St. Louis) or as 250,000 MW polymers for sugar beets(Fluka, Switzerland). The bacteria produce the polymer as a source ofstored nutrient. The fermentation of these polymers by bacteria wasdeveloped in the 1970s by ICI in the UK and Solvay et Cie in Belgium, asa potentially biodegradable plastic for tampon covers and other uses.The system responsible for the synthesis of the poly D-β-hydroxybutyratehas now been cloned and variations in the composition of the polymerproduced, based on the substrates given to the bacteria demonstrated.However, these polymers failed to be able to compete with petroleumbased plastics. Nevertheless the genes responsible for the synthesis ofpolyalkanoates has been cloned and expressed in a number ofmicro-organisms (93, 102, 113) allowing for production of this materialin a variety of organisms under extremely variable conditions.

[0054] Poly D-β-hydroxybutyrate comes in a number of forms fromdifferent biological sources as an insoluble white powder with littletaste and no odour and is suitable for incorporation into compositionsfor oral or other means of administration. Esterases capable of breakingthe ester bonds of this material are ubiquitous in plasma and mostcells. These polymer are also easily split by alkaline hydrolysis invitro to make a series of polymers culminating in the production of themonomer of MW 104, which is transported from gut to portal vein by thenormal monocarboxylate transporter. Alternatively acid hydrolysis may becarried out using the published method referred to in the Flukapromotional material.

[0055] Preferred forms of D-β-hydroxybutyrate polymer are oligomers ofthat ketone body designed to be readily digestable and/or metabolised byhumans or animals. These preferably are of 2 to 100 repeats long,typically 2 to 20 and most conveniently from 3 to 10 repeats long. Itwill be realised that mixtures of such oligomers may be employed withadvantage that a range of uptake characteristics might be obtained.

[0056] Particularly preferred are cyclic oligomers ofD-β-hydroxybutyrate, known as oligolides, having formula

[0057] where n is an integer of 1 or more

[0058] or a complex thereof with one or more cations or a salt thereof

[0059] Preferred cations are sodium, potassium, magnesium and calcium.Such cations are typically balanced by a physiologically acceptablecounter-anion such that a salt is provided.

[0060] Examples of typical physiologically acceptable salts will beselected from sodium, potassium, magnesium, L-Lysine and L-arginine oreg. more complex salts such as those of methyl glucamine salts

[0061] Preferably n is an integer from 1 to 200, more preferably from 1to 20, most preferably from 1 to 10 and particularly conveniently is 1,ie. (R, R, R)-4,8,12-trimethyl-1, 5,9-trioxadodeca-2,6,10-trione, 2, 3,4 or 5.

[0062] Cyclic oligomers for use in the invention may be provided, interalia, by methods described by Seebach et al. Helvetia Chimica Acta Vol71 (1988) pages 155-167, and Seebach et al. Helvetia Chimica Acta, Vol77 (1994) pages 2007 to 2033. For some circumstances such cyclicoligomers of 5 to 7 or more (R)-3-hydroxybutyrate units may be preferredas they may be more easily broken down in vivo. The methods of synthesisof the compounds described therein are incorporated herein by reference.

[0063] In preferred forms of all of the aspects of the invention, wherethe oligomer of of D-β-hydroxybutyrate does not include acetoacetylgroups it is optionally and preferably administered together with aphysiological ratio of acetoacetate or a metabolic precursor ofacetoacetate.

[0064] Once the monomer is in the blood stream and since liver isincapable of metabolizing ketone bodies but can only alter the ratio ofD-β-hydroxybutyrate/acetoacetate, the ketone bodies are transported toextrahepatic tissues where they can be utilized. The blood levels ofketones achieved are not be subject to variation caused by noncompliantingestion of carbohydrate, as is the case with the present ketogenicdiet. Rather, they would simply be an additive to the normal diet, givenin sufficient amounts to produce a sustained blood level typically ofbetween 0.3 to 20 mM, more preferably 2 to 7.5 mM, over a 24 hourperiod, depending upon the condition being treated. In the case ofresistant childhood epilepsy, blood levels of 2 mM are currently thoughtto be sufficient. In the case of Alzheimer's disease, attempts could bemade to keep levels at 7.5 mM achieved in the fasting man studies, in aneffort to provide alternative energy and acetyl CoA supplies to braintissue in Alzheimer's patients where PDH capacity is impaired because ofexcess amounts of Aβ₁₋₄₂ amyloid peptide (77, 78).

[0065] The determination by the inventor that D-β-hydroxybutyrate andits mixtures with acetoactetate act as a nerve stimulant, eg. nervegrowth stimulant and/or stimulant of axon and dendritic growth, opens upthe option of raising ketone body levels to lesser degrees than requirednutritionally in order to treat neurodegeneration.

[0066] Compositions of the invention are preferably sterile and pyrogenfree, particularly endotoxin free. Secondly, they are preferablyformulated in such a way that they can be palatable when given as anadditive to a normal diet to improve compliance of the patients intaking the supplements. The oligomers and polymers are generally tasteand smell free. Formulations of D-β-hydroxybutyrate and its mixtureswith acetoacetate may be coated with masking agents or may be targetedat the intestine by enterically coating them or otherwise encapsulatingthem as is well understood in the pharmaceuticals art.

[0067] Since ketone bodies contain about 6 calories/g, there ispreferably a compensatory decrease in the amounts of the other nutrientstaken to avoid obesity.

[0068] Particular advantages of using the ketone bodies or precursorssuch as poly or oligo-D-β-hydroxybutyrate or acetoacetate esters are:

[0069] 1) they can be eaten with a normal dietary load of carbohydratewithout impairing its effects.

[0070] 2) they will not raise blood VLDL, as with current cream andmargarine containing diets, thus eliminating the risk of acceleratedvascular disease, fatty liver and pancreatitis,

[0071] 3) they will have a wider range of use in a greater variety ofpatients, including: type II diabetes to prevent hypoglycemic seizuresand coma, in Alzheimer's disease and other neurodegenerative states toprevent death of nerve cells eg. hippocampal cells, and in refractoryepilepsy due to either decreases in cerebral glucose transporters,defects in glycolysis, or so called Leigh's syndromes with congenitaldefects in PDH.

[0072] The second group of particular alternatives are acetoacetateesters of D-β-hydroxybutyrate. Esters which provide a physiologicalratio of acetoacetate to D-β-hydroxybutyrate are preferred eg. from 1:1to 1:20, more preferably from 1:1 to 1:10. The tetramer ofD-β-hydroxybutyrate with a terminal acetoacetate residue is particularlypreferred. Such materials have the added virtue of having aphysiological ratio of D-β-hydroxybutyrate/acetoacetate moieties, thusremoving the burden on liver of having to adjust the redox state of theadministered nutrient without inducing abnormal reduction of hepatic[NAD⁺]/[NADH] as occurs with excessive alcohol consumption. Thepolymeric esters, depending upon their length, have decreasing watersolubility, but are heat stable. Such polymers can for example be usedin oral and parenteral use in emulsions, whereas acetoacetate, in theunesterified state, is less preferred as it is subject to spontaneousdecarboxylation to acetone with a half time at room temperature of about30 days.

[0073] Examples of poly D-β-hydroxybutyrate or terminally oxidized polyD-β-hydroxybutyrate esters useable as ketone body precursors are givenbelow.

[0074] Poly (R) 3-Hydroxybutyric acid

[0075] In each case n is selected such that the polymer or oligomer isreadily metabolised on administration to a human or animal body toprovide elevated ketone body levels in blood. Preferred values of n areinetegers of 0 to 1,000, more preferably 0 to 200, still more preferably1 to 50 most preferably 1 to 20 particularly conveniently being from to5.

[0076] A number of variations of this material, including the polyesterD-β-hydroxybutyrate itself can also be tried for suitable manufacturingcharacteristics. The material is a tasteless white powder. After partialalkaline hydrolysis, a mixture of varying chain length polymers would beprovided, which would tend to smooth gut absorption and maintain highsustained levels of ketone over a 24 hour period.

[0077] Treatment may comprise provision of a significant portion of thecaloric intake of patients with the D-β-hydroxybutyrate polyesterformulated to give retarded release, so as to maintain blood ketones inthe elevated range, eg. 0.5 to 20 mM, preferably 2-7.5 mM, range over a24 hour period. Release of the ketone bodies into the blood may berestricted by application of a variety of techniques such asmicroencapsulation, adsorption and the like which is currently practisedin the oral administration of a number of pharmaceutical agents.Enetrically coated forms targeting delivery post stomach may beparticularly used where the material does not require hydrolysis in acidenvironment. Where some such hydrolysis is desired uncoated forms may beused. Some forms may include enzymes capable of cleaving the esters torelease the ketone bodies such as those referred to in Doi. MicrobialPolyesters.

[0078] Intravenous infusion of sodium salts of D-β-hydroxybutyrate hasbeen performed on normal human subjects and patients for a number ofconditions, eg. those undergoing treatment for severe sepsis in anintensive care unit. It was found to be non-toxic and capable ofdecreasing glucose free fatty acids and glycerol concentration, butineffective in decreasing leucine oxidation.

[0079] The monomer of D-β-hydroxybutyrate is a white, odourless crystalwith a slightly tart or acid taste which is less in intensity incomparison to vinegar or lemon juice. It can be formulated into mostfoodstuffs, eg. drinks, puddings, mashed vegetables or inert fillers.The acid forms of D-β-hydroxybutyrate are suitable for use orally asthey have a pKa of 4.4. This is less acid than citric acid with pKa1 of3.1 and pKa2 of 4.8 and slightly more acidic than acetic acid with a pKaof 4.7.

[0080] Preferably, only the natural D- or (R) isomer is used in thisformulation. Since in practice it is not possible to achieve absoluteisomeric purity, the article of commerce currently sold by Sigma. St.Louis Mo. or Fluka, Ronkonkoma, N.Y. is the most suitable for thispurpose. The optical rotation of the commercially availableD-β-hydroxybutyratic acid is −25°±1 at the wavelength of Na and itsmelting point 43-46° C. The optical rotation of the Na salt ofD-β-hydroxybutyrate is −14.5° and its melting point 149-153° C. Both canbe assayed by standard enzymatic analysis using D-β-hydroxybutyratedehydrogenase (EC 1.1.1.30)(5). Acetoacetate can be determined using thesame enzyme (56). The unphysiological (S) isomer is not measurable withenzymatic analysis but can be measured using GC mass spec (13).

[0081] For a 1500 calorie diet, the human adult patient could consume198 g of ketones per day. For a 2000 calorie diet of the sameproportions, one could consume 264 g of ketones per day. On theketogenic lipid diet blood ketones are elevated to about 2 mM. On theketone diet, ketone levels should be higher because ketones have beensubstituted at the caloric equivalent of fat, that is 1.5 g of ketone/1g of fat. Accordingly, blood ketones should be approximately 3 mM, butstill below the level achieved in fasting man of 7.5 mM.

[0082] The advantage of using ketone bodies themselves are several.Firstly, provision of ketone bodies themselves does not require thelimitation of carbohydrate, thus increasing the palatability of thedietary formulations, particularly in cultures where high carbohydratediets are common. Secondly, ketone bodies can be metabolized by muscle,heart and brain tissue, but not liver. Hence the fatty liver, which maybe an untoward side effect of the ketogenic diet, is avoided. Thirdly,the ability to include carbohydrate in the dietary formulationsincreases the chance of compliance and opens up practical therapeuticapproaches to type II diabetics where insulin is high, making the knownketogenic diet unworkable.

[0083] The present inventor has determined that, while any elevation ofketone bodies may be desirable, a preferred amount of ketone bodies tobe administered will be sufficient to elevate blood levels to the 0.5 to20 mM level, preferably to the 2 mM to 7.5 mM level and above,particularly when attempting to arrest the death of brain cells indiseases such as Alzheimer's. While dead cells cannot be restored,arrest of further deterioration and at least some restoration offunction is to be anticipated.

[0084] Thus in a first aspect of the present invention there is providedthe use acetoacetate. D-β-hydroxybutyrate or a metabolic precursor ofeither in the manufacture of a medicament or nutritional aid (i) forincreasing cardiac efficiency, particularly efficiency in use of glucose(ii) for providing energy source, particularly in treating diabetes andinsulin resistant states or by increasing the response of a body toinsulin (iii) for reversing, retarding or preventing nerve cell damageor death related disorders, particularly neurodegenerative disorderssuch as memory associated disorders such as Alzheimer's, seizure andrelated states such as encepalophies such as CJD and BSE.

[0085] The term metabolic precursor thereof particularly relates tocompounds that comprise 1,3-butandiol, acetoacetyl orD-β-hydroxybutyrate moieties such as acetoacetyl-1,3-butandiol,acetoacetyt-D-β-hydroxybutyrate, and acetoacetylglycerol. Esters of anysuch compounds with monohydric, dihydric or trihydric alcohols is alsoenvisaged.

[0086] This aspect includes such use as a neuronal stimulant eg capableof stimulating axonal and/or dendritic growth in nerve cells, eg. inHippocampus or Substantia nigra particularly in diseases whereneurogeneration has serious clinical consequences.

[0087] In diabetic patients this use of these compounds allowsmaintenance of low blood sugar levels without fear of hypoglycemiccomplications. In normal non-diabetic subjects the fasting blood sugaris 80 to 90 mg % (4.4-5 mM) rising to 103 mg % (7.2 mM) after a meal. Indiabetics ‘tight control’ of diabetes has long been recommended as amethod for retardation of vascular complications but, in practice,physicians have found it difficult to keep blood sugars tightlycontrolled below 150 mg % (8.3 mM) after eating because of hypoglycaemicepisodes. Hypoglycaemic coma occurs regularly in normal subjects whoseblood sugar drops to 2 mM. As discussed earlier, (62, 63) in thepresence of 5 mM blood ketones there are no neurological symptoms whenblood sugars fall to below 1 mM.

[0088] The present inventor has determined that supplementing type IIdiabetics with ketone bodies would allow better control of blood sugar,thus preventing the vascular changes in eye and kidney which occur nowafter 20 years of diabetes and which are the major cause of morbidityand mortality in diabetics.

[0089] Where the therapy is aimed at seizure related disorders, such asrefractory epilepsy as is treated by the ketogenic diet, therapy isimproved by use of ketone bodies, their polymers or esters or precursorssuch as butandiol compounds, due to the reduction or elimination of highlipid and carbohydrate content. Such patients include those with geneticdefects in the brain glucose transporter system, in glycolysis or in PDHitself such as in Leigh's syndrome.

[0090] Particular disorders treatable with these medicaments areapplicable to all conditions involving PDH blockage, including thoseconditions occuring after head trauma, or involving reduction oreleimination of acetyl CoA supply to the mitochondrion such as insulincoma and hypoglycaemia, defects in the glucose transporter in the brainor in glycolytic enzyme steps or in pyruvate transport.

[0091] Preferably the hydroxybutyrate is in the form of non-racemicD-β-hydroxybutyrate and more preferably it is administered in a formwhich also supplies acetoacetate. Preferably the metabolic precursor isone which when administered to a human or animal body is metabolised eg.by liver, to produce one or both of D-β-hydroxybutyrate andacetoacetate, more preferably in a physiological ratio. Particularlypreferred are poly-D-β-hydroxybutyric acid oracetoacetoyl-β-hydroxybutyrate oligomers or an ester of one or both ofthese. Lower alkyl esters such as C₁₋₄ alkyl esters may be employed butmore preferably are more physiologically acceptable esters such as therespective 1,3-butandiol esters, particularly employing(R)-1,3-butandiol. Most preferred are the acetoacetyl-tri-, tetra- andpenta-D-β-hydroxybutyrate esters. Ester precursors will include estersof 1,3-butandiol, preferably (R) form and particularly acetoacetateesters such as acetoacetyl glycerol.

[0092] Preferred poly D-β-hydroxybutyrate esters are those which areesters of the preferred oligomers of 2-100 repeats, eg. 2-20 repeatsmost preferably 2-10 repeats.

[0093] Where the medicament or nutritional product of the invention isfor use without prolonged storage it is convenient to use it in the formof a liquid or solid composition comprising the hydroxy substitutedcarboxylic acid and/or the ketone, preferably comprising both and wherethese are the D-β-hydroxybutyrate acids and acetoacetate togetherpreferably in the ratio of about 3:1 to 5:1, more preferably about 4:1.

[0094] Where the medicament or aid comprises acetoacetate it ispreferably not stored for a prolonged period or exposed to temperaturesin excess of 40° C. Acetoacetate is unstable on heating and decomposesviolently at 100° C. into acetone and CO₂. In such circumstances it ispreferred that acetoacetate is generated by the composition on contactwith the bodies metabolic processes. Preferably the compositioncomprises an ester precursor of actetoacetate. For example, the ethylester of acetoacetate is relatively stable with a boiling point of180.8° C.

[0095] Still more preferably, the medicament or aid comprises anacetoacetyl ester of D-β-hydroxybutyrate or such an ester of an oligomerof D-β-hydroxybutyrate as described. This may be supplemented withD-β-hydroxybutyrate or one of the polymers of that e.g.oligo-D-β-hydroxybutyrate, in order to bring about the preferred ratioof the two components. Such a composition will provide the two preferredcomponents when the ester and polymer are metabolised in the stomach orin the plasma of the human or animal which has consumed them. Again an(R) 1,3-butandiol ester of the acetoacetyl-D-β-hydroxybutyrate may bemost preferred as it will be more lipophilic until metabolised orotherwise deesterified and all its components are convereted to thedesired ketone bodies.

[0096] A second aspect of the invention provides novel esters ofacetoacetate for use in therapy or as a nutritional aid. Such esters mayinclude C₁₋₄ alkyl esters but most preferred are theD-β-hydroxybutyryl-acetoacetate esters referred to above.

[0097] A third aspect of the present invention provides apoly-D-β-hydroxybutyrate for use in therapy, particularly where this isin a form selected for its ability to be degraded in acid conditions ofthe stomach or by esterases in vivo.

[0098] A fourth aspect of the invention provides a method for thesynthesis of D-β-hydroxybutyryl-acetoacetate esters comprising thereaction of acetoacetic acid halide, e.g. acetoacetyl chloride, withD-β-hydroxybutyrate. Preferably this is achieved by reacting acetoaceticacid with an activating agent, such as thionyl chloride, to produce theacid chloride.

[0099] A fifth aspect of the present invention provides a method for thesynthesis of D-β-hydroxybutyryl-acetoacetate esters comprising thereaction of D-β-halobutyrate or its oligomers with acetoacetic acid,activated forms thereof or diketene.

[0100] A sixth aspect of the present invention provides aD-β-hydroxybutyryl-acetoacetate ester per se, a physiologicallyacceptable salt or short or medium chain mono, di or trihydric alcoholor 1,3-butandiol estsr thereof.

[0101] A seventh aspect of the present invention providespoly-D-β-hydroxybutyrate together with a pharmaceutically orphysiologically acceptable carrier.

[0102] An eighth aspect of the present invention provides a compositioncomprising D-β-hydroxybutyrate and acetoacetic acid in a ratio of from1;1 to 20:1, more preferably 2:1 to 10:1 and most preferably from 3:1 to5:1 together with a pharmaceutically or physiologically acceptablecarrier. Preferably the ratio of these components is about 4:1. Such acomposition does not consist of plasma, serum or animal or plant tissuealready used as a medicament or foodstuff, thus it is that which ispreferably sterile and pyrogen free. Particularly the ketione bodiescomprise at least 5% of the composition by weight, more preferably 20%or more and most preferably 50% to 100%. The composition may be adaptedfor oral, parenteral or any other conventional form of administration.

[0103] A ninth aspect of the present invention comprises a method oftreating a human or animal in order to increase their cardiac efficiencycomprising administering to that person at least one of a materials foruse in the first to eight aspects of the invention.

[0104] A tenth aspect of the present invention comprises a method oftreating a human or animal in order to increase their the response toinsulin comprising administering to that person at least one of amaterials for use in the first to eight aspects of the invention.

[0105] An eleventh aspect of the present invention comprises a method oftreating a human or animal in order to treat an insulin resistant statecomprising administering to that person least one at least one of amaterials for use in the first to eight aspects of the invention.

[0106] By insulin resistant state herein is included forms of diabetes,particularly those that do not respond fully to insulin.

[0107] A twelvth aspect of the invention provides a method of treating ahuman or animal in order to treat a nerve cell, eg. brain cell, death ordamage related disorder as referred to for the first aspect,particularly a neurodegenerative disorder eg. such as those related toneurotoxic conditions such as presence of amyloid protein, eg. a memoryassociated disorder such as Alzheimer's disease, or epileptic seizures,comprising administering to that person least one at least one of amaterials for use in the first to eight aspects of the invention.

[0108] Preferred methods of the ninth to twelvth aspects of theinvention use the preferred ketones and polyacids and acid esters of theinvention.

[0109] Methods of preparing poly D-β-hydroxybutyrate are notspecifically claimed as these are known in the art. For example Shang etal. (1994) Appli. Environ. Microbiol. 60: 1198-1205. This polymer isavailable commercially from Fluka Chemical Co. P1082, cat#81329,1993-94, 980. Second St. Ronkonkoma N.Y. 11779-7238, 800 358 5287.

[0110] Particular advantages of use of the biologically availablepolymers of the invention include the reduction in the amount of counterions such as sodium that have to be coadministered with them. Thisreduction in sodium load is advantageous particularly in ill health. Bybiologically available is meant those materials which can be used by thebody to produce the least one of a D-β-hydroxybutyrate, acetoacetate anda mixture of these in physiological ratio as described above

[0111] The amount of ketone bodies used in treatment ofneurodegeneration such as Alzheimer's and Parkinsonism will preferablyelevate blood levels to 0.5 mM to 20 mM, eg 2 mM to 7.5 mM as describedabove. The present inventor estimates that 200 to 300 g (0.5 pounds) ofketone bodies per patient per day might be required to achieve this.Where the treatment is through maintenance of cells against the effectsof neurotox in this may be at a higher level, eg. 2 to 7.5 mM in blood.Where it relies on the nerve stimulatory factor effect of theD-β-hydroxybutyrate so produced the amount administered nay be lower,eg. to provide 0.2 to 4 mM, but can of course be more for this or otherdisease.

[0112] It will be realised that treatment for neurodegenerative diseasessuch as Alzheimer's will most effectively be given soon afteridentifying patient's with a predisposition to develop the disease. Thustreatment for Alzheimers' most effectively follows a positive testresult for one or more conditions selected from the group (i) mutationsin the amyloid precursor protein gene on chromosome 21, (ii) mutationsin the presenilin gene on chromosome 14, (iii) presence of isoforms ofapolipoprotein E. Other tests shown to be indicative of Alzheimer's willof course be applicable.

[0113] Following such a positive test result it will be appropriate toprevent the development of memory loss and/or other neurologicaldysfunction by elevation of the total sum of the concentrations of theketone bodies D-β-hydroxybutyrate and acetoacetate in the patient'sblood or plasma to say between 1.5 and 10 mM, more preferably 2 to 8 mM,by one of several means. Preferably the patient is fed a diet ofsufficient quantities of D-β-hydroxybutyrate, its metabolisablepolymers, its acetoacetate esters or their precursors (R)-1,3-butandioland its acetoacetate esters, eg. acetoacetyl glycerol, or itsadministered intravenously or intrarterially the ketone bodiesD-β-hydroxybutyrate and acetoacetic acid. All of the organic materialsreferred to above are optionally in salt or ester form. Examples oftypical physiologically acceptable salts will be selected from sodium,potassium, magnesium, L-Lysine and L-arginine or eg. more complex saltssuch as those of methyl glucamine salts. Esters will be those asdescribed previously for other aspects of the invention.

[0114] A still further aspect of the invention provides the ketonebodies of the invention by suitable control of diet. Thus this aspectprovides a method of treatment of a human or animal for a disorder ofone or more of the ninth to the twelvth aspects of the inventioncomprising one of (i) total fasting of the individual and (ii) feedingthe individual a ketogenic diet eg. of 60-80% lipid with carbohydratecontent 20% or less by weight.

[0115] For the purpose of treaing seizures, eg. in epilepsy, a diet mayinvolve ad lib ingestion of carbohydrate by oral or enteral route or ofthe compounds specified above.

[0116] In all these treatments other than the ketogenic diet there isthe improvement that a method of avoiding drop in blood ketones whichaccompanies the ingestion of excess carbohydrate and a method whichavoids feeding of excess lipid which accelerates the synthesis by liverof fatty acids and cholesterol which would otherwise contribute tovascular disease.

[0117] It will be realised that hypoglycemic brain dysfunction will alsobe treatable using the treatments and compositions and compounds of thepresent invention. A further property associated with the presenttreatment will be general improvement in muscle performance.

[0118] The provision of ketone body based foodstuffs and medicaments ofthe invention is faciliated by the ready availability of a number ofrelatively cheap, or potentially cheap, starting materials from which(R)-3-hydroxybutyric acid may be derived (see Microbial PolyestersYoshiharu Doi. ISBN 0-89573-746-9 Chapters 1.1, 3.2 and 8). Theavailability of genes capable of insertion into foodstuff generatingorganisms provides a means for generating products such as yoghurts andcheese that are enriched in either poly-(R)-3-hydroxybutyric acid or,after breakdown with enzymes capable of cleaving such polymers, with themonomeric substance itself (see Doi. Chapter 8).

[0119] The present invention will now be described further by way ofillustration only by reference to the following Figures and experimentalexamples. Further embodiments falling within the scope of the inventionwill occur to those skilled in the art in the light of these.

FIGURES

[0120]FIG. 1 is a graph showing blood (R)-3-hydroxybutyrate levelproduced after time after gavage of (R)-3-hydroxybutyrate, an oligomerof this as produced in Example 1 and an acetoaceryl monomer therof asproduced in Example 2.

[0121]FIG. 2 is a graph showing blood (R)-3-hydroxybutyrate levelproduced after time after feeding rats with the triolide of(R)-3-hydroxybutyrate a cyclic oligomer produced in Example 1 in voghurtand controls fed yoghurt alone.

EXAMPLES Example 1

[0122] Preparation of Oligomers of (R)-3-hydroxybutyric Acid(D-β-hydroxybutyrate).

[0123] (R)-3-hydroxybutyric acid (Fluka-5.0 g: 0.048 mole), p-toluenesulphonic acid (0.025 g) and benzene (100 ml) were stirred under refluxwithn a Dean-Stark trap arrangement for 24 hours. The reaction mixturewas cooled and the benzene evaporated in vacuo (0.5 mm Hg). 4.4 g ofcolourless oil was obtained of which a 20 mg sample was converted to themethyl ester for analysis of number of monomer repeats using NMR. Thesestudies show that the product is a mixture of oligomers ofD-β-hydroxybutyrate of avaerage number of repeats 3.75, being mainly amixture of trimers, tetramers and pentamers with the single mostabundant material being the tetramer. The product mixture was soluble in1 equivalent of sodium hydroxide.

Example 2

[0124] Preparation of Acetoacetyl Ester of Oligomeric(R)-3-hydroxybutyric Acid.

[0125] A further batch of the colourless oil product from Example 1 (4.5g) was heated for 1 hour at 60° C. with diketene (3.8 g) and sodiumactetate (0.045 g) under nitrogen. Further diketene (3.8 g) was addedand the reaction heated for a further hour, cooled and diluted withether, washed with water and then extracted with saturated sodiumbicarbonate (5×100 ml). Combined extract was washed with ether thenacidified with concentrated HCl (added dropwise). Ethyl acetateextraction (3×50 ml) was followed by drying over magnesium sulphate andevaporation in vacuo. A yellow solid/oil mixture was obtained (7.6 g)which was chromatographed on a silica column usingdichloromethane/methanol (98:2) to give a light amber oil product.Faster moving impurities were isolated (1.6 g) and after recolumningcarbontetrachloride/methanol (99:1) 0.8 g of oil was recovered which wasshown by NMR and Mass spectrometry to be the desired mixture ofacetoacetylated oligomers of D-β-hydroxybutyrate. The product mixturehad an Rf of 0.44 in dichloromethane/methanol (90:1) and was soluble in1 equivalent of sodium hydroxide. Both products of Example 1 and Example2 are susceptible to separation of individual components by preparativeHPLC.

Example 3

[0126] Oral Administration of D-β-hydroxybutyrate, Oligomers andAcetoacetyl D-β-hydroxybutyrate Oligomers to Rats.

[0127] The ability of orally administered D-β-hydroxybutyrate and theoligomers of Examples 1 and 2 to raise blood ketone body levels wasinvestigated as follows. Rats were starved overnight and then gavagedwith 100 μl/100 g bodyweight of 4M D-β-hydroxybutyrate brought to pH7.74 using methyl glucamine. Blood levels of D-β-hydroxybutyratemeasured using and NAD+/EDTA assay of Anal. Biochem. 131, p478-482(1983). 1.0 ml of a solution made up from 2-amino-2-methyl-1-propanol(100 mM pH 9.9, 0.094 g/10 ml), NAD+ (30mM, 0.199 g/10 ml) and EDTA (4mM, 0.015 g/10 ml) was added to each of a number of cuvettes and 4 μlsample or D-β-hydroxybutyrate control.

[0128] As the rats had been fasted the initial levels ofD-β-hydroxybutyrate were elevated from the 0.1 mM fed state. However,consistent serum increases of D-β-hydroxybutyrate, between 1 and 3.2 mMincrease in each case, were provided.

[0129] This procedure was repeated with 2M solutions of the mixtures ofD-β-hydroxybutyrate oligomers and their acetoacetyl esters described inExamples 1 and 2. The D-β-hydroxybutyrate oligomer (19/1 in FIG. 1) andthe acetoacetyl ester (20/4 in FIG. 1) were both brought to pH 7.6 withmethyl glucamine and the blood D-β-hydroxybutyrate level monitored usingthe aforesaid assay procedure. Increases in serum D-β-hydroxybutyratewere shown to be of 0.5 to 1.2 mM at 60 and 120 minutes after gavaging.These results demonstrate the efficacy of orally administeredD-β-hydroxybutyrate and its metabolic precursors of the invention inraising blood levels significantly for a period of hours after intake.

[0130] It was noted that the oligomeric esters 19/1 and 20/4, while notelevating the blood ketone body level as high as the monomer itself, didresult in elevation for a much longer period of time and thus are suitedto adminsitration less frequently than the monomer.

Example 4

[0131] TABLE 2 Sample 1500 calorie ketogenic diet using ketone bodies,their esters or polymers. The ketones were assumed to contain 6 kcal/g,fats 9 kcal/g, carbohydrate and protein 4 kcal/g. Ketones have beensubstituted to give equivalent calories. Pro- CHO Amount (g) Fat (g)tein (g) (g) Ketones (g) Breakfast Egg 32 4 4 apple juice 70 7 ketones66 66 skim milk 92 0 2 3 Total Breakfast 4 6 10 66 Lung Lean beef 121.75 3.5 cooked carrots 45 0.6 3 canned pears 40 4 ketones 69.75 69.75skim milk 92 2 3 Total Lunch 1.75 6.1 10 69.75 Supper Frankfurter 22.5 63 cooked broccoli 50 1 2 watermelon 75 5 ketones 62.25 62.25 skim milk92 2 3 Total Supper 6 6 10 62.25 Daily Total 11.75 18.1 30 198

Example 5

[0132] Effect of Increased Blood D-β-hydroxybutyrate Levels on WholeBrain GABA Levels.

[0133] To assess the effect of D-β-hydroxybutyrate on whole brain GABAlevels, and thus provide an indication of antiepileptic effect of ketonebody or precursor treatment aimed at increasing blood ketone bodylevels, whole rat brain was frozen at set times after administration ofD-β-hydroxybutyrate as described in Example 3. GABA was assayed usingstandard HPLC technique and related to protein content using standardprotein assay. At t=0 GABA levels were 191 pmoles/μg protein while at120 minutes this was elevated at 466 pmoles/μgprotein, demonstratingantepileptic potential.

Example 6

[0134] Effect of D-β-hydroxybutyrate on β-amyloid Toxicity toHippocampal Cells in Vitro

[0135] Culture Medium and Chemicals

[0136] The serum free medium used from 0 to day 4 contained Neurobasalmedium with B27 supplement diluted 50 fold (Life Technology,Gaithersburg, Md.) to which was added: 0.5 mM L-glutamine, 25 μM NaL-glutamate, 100 U/ml penicillin and 100 μg/ml streptomycin. After day4, DMEM/F12 medium containing 5 μM insulin, 30 nM 1-thyroxine, 20 nMprogesterone, 30 nM Na selenite 100 U/ml penicillin and 100 μg/mlstreptomycin were used.

[0137] Hippocampal Microisland Cultures

[0138] The primary hippocampal cultures were removed from Wistar embryoson day 18 and dispersed by gentle aggitation in a pipette. Thesuspension was centrifuge at 1,500×g for 10 min and the supernatantdiscarded. New media was make 0.4-0.5×10⁶ cells/ml. Ten μl of thissuspension was pipetted into the center of poly D-lysine coated culturewells and the plates incubated at 38° C. for 4 hrs and then 400 μl offresh Neurobasal media was added. After 2 days of incubation, half ofthe media was exchanged for fresh media and the incubation continued for2 more days. After day 4, the medium was changed with DMEM/F12 mediumcontaining 5 μM insulin, 30 nM 1-thyroxine, 20 nM progesterone, 30 nM Naselenite 100 U/ml penicillin and 100 μg/ml streptomycin. The wells weredivided into 4 groups: half the wells received Na D-β-hydroxybutyrate toa final concentration of 8 mM while and half of the wells received 5 nMamyloid β₁₋₄₂ (Sigma). These media were exchanged 2 days later (day 8)and the cells were fixed on day 10 and stained with anti MAP2(Boehringer Manheim, Indianapolis Ind.) to visual neurons and vimentinand GFAP (Boehringer) to visualize glial cells.

[0139] Results

[0140] Cell Counts

[0141] Addition of D-β-hydroxybutyrate to the incubation resulted in anincrease in the neuronal cell number per microisland from a mean of 30to mean of 70 cells per microisland. Addition of 5 nM amyloid β₁₋₄₂ tothe cultures reduced the cell numbers from 70 to 30 cells permicroisland, confirming the previous observations of Hoshi et al, thatamyloid β₁₋₄₂ is toxic to hippocampal neurons. AdditionD-β-hydroxybutyrate to cultures containing amyloid β₁₋₄₂ increased thecell number from a mean of 30 to 70 cells per microisland. From thesedata we conclude that addition of substrate level quantities ofD-β-hydroxybutyrate, to media whose major nutrients are glucose,pyruvate and L-glutamine, slows the rate of cell death in culture. Wefurther conclude that D-β-hydroxybutyrate can decrease the increasedrate of hippocampal cell death caused by the addition of amyloid β₁₋₄₂in culture.

[0142] The number of dendritic outgrowths and the length of axons wereboth observed to have increased with presence of D-β-hydroxybutyrate,whether β₁₋₄₂ was present or not. This is indicative of nerve growthfactor like behaviour.

Example 7

[0143] Preparation of(R,R,R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione: triolide of(R)-3-hydroxybutyric acid.

[0144] Synthesis was as described in Angew. Chem. Int. Ed. Engl. (1992),31, 434. A mixture of poly[(R)-3-hydroxybutyric acid] (50 g) andtoluene-4-sulphonic acid monohydrate (21.5 g, 0.113 mole) in toluene(840 ml) and 1,2-dichloroethane (210 ml) was stirred and heated toreflux for 20 hours. The water was removed by Dean-stark trap for 15hours whereafter the brown solution was cooled to room temperature andwashed first with a half saturated solution of sodium carbonate thenwith saturated sodium chloride, dried over magnesium sulphate andevacuated in vacuo. The brown semi-solid residue was distilled using aKugelrohr apparatus to yield a white solid (18.1 g) at 120-130° C./0.15mmHg. Above 130° C. a waxy solid began to distill—distillation beingstopped at this point. The distilled material had mp 100-102° C.(literature mp 110-110.5° C.). Recrystallisation from hexane gavecolourless crystals in yield 15.3 g, Mp=107-108° C.; [α]_(D)-35.1(c=1.005, CHCl₃), (lit.=−33.9). ¹H NMR (300 MHz, CDCl₃): δ=1.30 (d, 9H,CH₃); 2.4-2.6 (m,6H; CH₂); 5.31-5.39 (M, 3H; HC—O). ¹³C NMR (CDCl₃)δ=20.86 (CH₃), 42.21 (CH₂), 68.92 (CH), 170.12 (CO). Elemental analysis:calculated for C₁₂H₁₈O₆: C, 55.81; H 7.02; Found: C, 55.67; H, 7.15.

Example 8

[0145] Oral Administration of Triolide of D-β-hydroxybutyrate of Example1 to Rats.

[0146] The ability of orally administered triolide to raise blood ketonelevels was investigated as follows. The day before the experimentcommenced, 12 Wistar rats weighing 316±10 g were placed in separatecages. They had no access to food for 15 hours prior to presentationwith triolide containing compositions, but water was provided adlibitum.

[0147] On the morning of the experiment 0.64 g of triolide was mixedwith 5 g Co-op brand Black Cherry yoghurt in separate feeding bowls for9 of the rats. The remaining 3 rats were given 5 g of the yoghurtwithout the triolide as controls. The yoghurt containing bowls wereplaced in the cages and the rats timed while they ate. Two of the threecontrol rats ate all the yoghurt and four of the six triolide yoghurtrats ate approximately half the provided amount. The remaining six ratsslept.

[0148] Control rats (n=2) were killed at 60 and 180 minutes afteringestion of yoghurt while triolide fed rats were killed at 80, 140, 150and 155 minutes. Blood samples were taken for assay ofD-β-hydroxybutyrate. Brains were funnel frozen and later extracted inperchloric acid and extracts neutralized and assayed. Blood levels of(R)-3-hydroxybutyrate were measured using a NAD⁺/EDTA assay of Anal.Biochem (1983 ) 131, p478-482. 1.0 ml of a solution made up from2-amino-2-methyl-1-propanol (100 mM pH 9.9, 0.094 g/10 ml), NAD⁺ (30 mM,0.199 g/10 ml) and EDTA (4 mM, 0.015 g/10 ml) was added to each of anumber of cuvettes and 4 μl sample or D-β-hydroxybutyrate control.

[0149] The two control rats ate 5.2±0.01 g yoghurt and their plasma(R)-3-hydroxybutyrate concentrations were about 0.45 mM at 60 minutesand 180 minutes. The four triolide fed rats ate 0.39±0.03 g of thetriolide and 2.6±0.2 g, of yoghurt. Their plasma D-β-hydroxybutyrateconcentrations were 0.8 mM after 80 minutes and 1.1 mM for the groupsacrificed at about 150 minutes. All rats displayed no ill effects fromingestion of triolide.

[0150] Thee test rats thus showed increase in plasma D-β-hydroxybutyrateover at least 3 hours with no ill effects. It should be noted that twoother rats fed approximately 1.5 g triolide each in ‘Hob-Nob’ biscuitshowed no ill effects after two weeks.

[0151] For all examples given above, it should be noted that theincreased levels of (R)-3-hydroxybutyrate will also be mirrored inacetoacetate levels, not measured here, as there is a rapidestablishment of equilibrium between the two in vivo such thatacetoacetate levels will be between 40 and 100% of the(R)-3-hydroxybutyrate levels.

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1. A method of treating a patient for a neuro-degenerative disordercomprising administering to that patient a therapeutically effectiveamount of one or more of D-β-hydroxybutyric acid, acetoacetate, or ametabolic precursor or physiologically acceptable salt ofD-β-hydroxybutyric acid or acetoacetate, such as to elevate thepatient's blood level of ketone bodies, defined as the sum total ofD-β-hydroxybutyric acid and acetoacetate, to a therapeutic leveleffective to treat the disorder wherein when a metabolic precursor isadministered it is not hydroxybutyryl carnitine.
 2. A method of treatinga patient in order to treat a neuro-degenerative disorder comprisingadministering to that patient a therapeutically effective amount of atleast one of D-β-hydroxybutyric acid, acetoacetate, or a metabolicprecursor or physiologically acceptable salt of D-β-hydroxybutyric acidor acetoacetate, such as to elevate the patient's blood level of ketonebodies, defined as the sum total of D-β-hydroxybutyric acid andacetoacetate, to a therapeutic level effective to treat the disorderwherein the patient's blood level is elevated to from 0.3 mM to 20 mM.3. A method of treating a CNS cell, peripheral nerve cell, or otherwiseinsulin insensitive cell in need of therapy for one or more ofneuro-degeneration, GABA preventable seizure, or insufficient ability tometabolise glucose, comprising administering to that cell one or morecompounds selected from the group consisting of D-β-hydroxybutyric acid,acetoacetate, compounds which are oligomers of D-β-hydroxybutyric acid,acetoacetyl esters of D-β-hydroxybutyric acid and acetoacetyl esters ofoligomers of D-β-hydroxybutyric acid, and physiologically acceptablesalts thereof.
 4. A method of treating an patient for epilepsy, diabetesor an insulin resistant state comprising administering to that patient atherapeutically effective amount of one or more compounds selected fromthe group consisting of D-β-hydroxybutyric acid, acetoacetate andmetabolic precursors of D-β-hydroxybutyric acid or acetoacetate whichcomprise moieties selected from the group consisting of R-1,3-butandiol,acetoacetyl and D-β-hydroxybutyryl moieties and physiologicallyacceptable salts and esters thereof.
 5. A method as claimed in any oneof claim 1, claim 2, claim 3 and claim 4 wherein on administration ofthe compound to an unfasted patient in need of such therapy, the bloodlevel of ketone bodies, defined as the sum total of D-β-hydroxybutyricacid and acetoacetate, is raised to between 0.3 and 20 mM.
 6. A methodas claimed in claim 1 or claim 2 wherein the neurodegenerative disorderis selected from the group consisting of neurodegenerative disordersinvolving inability to metabolise glucose, memory loss in ageing,neurotoxic peptides or proteins, and genetic abnormality.
 7. A method asclaimed in claim 6 wherein the neurodegenerative disorder is selectedfrom those involving neurotoxic protein plaques.
 8. A method as claimedin claim 1 or claim 2 wherein the metabolic precursor is selected fromthe group consisting of Free Fatty Acids and compounds comprising1,3-butandiol, acetoacetyl or D-β-hydroxybutyryl moieties.
 9. A methodas claimed in claim 1, claim 2, claim 3 or claim 4 wherein the metabolicprecursor is a polymer or oligomer of D-β-hydroxybutyrate.
 10. A methodas claimed in claim 9 wherein the metabolic precursor is an acetoacetylester.
 11. A method as claimed in claim 9 wherein metabolic precursor isselected from the group consisting of compounds of general formulae

or physiologicial acceptable salts or esters thereof wherein in eachcase n is selected such that the polymer or oligomer is readilymetabolised on administration to a human or animal body to provideelevated ketone body levels in blood
 12. A method as claimed in claim 11wherein n is an integer of 0 to 1,000.
 13. A method as claimed in claim11 wherein n is an integer of from 1 to
 5. 14. A method as claimed inclaim 1, claim 2, claim 3 or claim 4 wherein the level of ketone bodiesproduced in the blood is in the ratio 1:1 to 20:1 of D-β-hydroxybutyrateto acetoacetate.
 15. A method as claimed in claim 9 wherein the oligomeris a cyclic oligomer of formula

where n is an integer of 1 or more or a complex thereof with one or morecations or a salt thereof
 16. A method as claimed in claim 15 whereinthe one or more cations are selected from the group consisting ofsodium, potassium, magnesium and calcium.
 17. A method as claimed inclaim 15 wherein n is an integer from 1 to to
 20. 18. A method asclaimed in claim 1 wherein it is (R, R,R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione.
 19. A compound offormula

or physiologicial acceptable salts or esters thereof. wherein n is aninteger from 0 to 1000
 20. A compound as defined in claim 19 wherein theester is selected from the group consisting of monohydric, dihydric ortrihydric alcohol esters
 21. A compound as claimed in claim 19 whereinthe ester is of (R)-1,3-butandiol.
 22. A compound as claimed in claim 19wherein n is selected from the group of integers 0, 1, 2, 3 and
 4. 23. Afoodstuff comprising poly D-β-hydroxybutyrate characterised in that itis derived from a foodstuff generating organism that has had a genecapable of producing D-β-hydroxybutyrate inserted therein.
 24. Afoodstuff charactersied in that it comprises at least 5% ketone bodiesby weight.
 25. A method for the synthesis ofD-β-hydroxybutyryl-acetoacetate or poly oroligo-D-β-hydroxybutyryl-acetoacetate esters comprising the reaction ofacetoacetic acid halide with D-β-hydroxybutyrate or poly- oroligo-D-β-hydroxybutyrate.
 26. A method for synthesis ofD-β-hydroxybutyryl-acetoacetate or oligo-D-β-hydroxybutyryl-acetoacetatecomprising reacting D-β-hydroxybutyryic acid with diketene.
 27. A methodof synthesising an oligomer of D-β-hydroxybutyric acid comprisingheating a solution of D-β-hydroxybutyric acid in a solvent until anoligomer of a desired number of repeats is produced.
 28. Use ofD-β-hydroxybutyric acid, acetoacetate, or a metabolic precursor orphysiologically acceptable salt of D-β-hydroxybutyric acid oracetoacetate for the manufacture of a medicament for the treatment of adisorder by a method as set out in any one of claims 1 to 14 providedthat when the use is of a metabolic precursor that is not racemichydroxybutyryl carnitine.
 29. A foodstuff as claimed in claim 23 orclaim 24 for use in therapy.
 30. Poly-D-β-hydroxybutyrate for use intherapy
 31. A composition comprising a compound selected from thoseclaimed in any one of claims 15 to 18 and poly D-β-hydroxybutyratetogether with a physiologically acceptable carrier, in sterile andpyrogen free form.